Reduction of endotoxin in polysialic acids

ABSTRACT

The present invention relates to process for reducing the endotoxin content of a sample of fermentation broth containing polysialic acid and endotoxin comprising the sequential steps: (i) adding to the sample a base having a pKa of at least 12 to form a basic solution having a pH of at least 12, incubating the solution for a pre-determined time at a pre-determined temperature; and (ii) recovery of PSA, suitably by (iii) passing the sample through an anion-exchange column whereby polysialic acid is absorbed on the ion exchange resin; (iv) washing the column with one washing buffer, whereby polysialic acid remains absorbed on the ion exchange resin; and (v) eluting the polysialic acid from the column using an elution buffer to provide a product solution of polysialic acid having reduced endotoxin content.

The present invention relates to the reduction of endotoxin inpolysialic acid (PSA) and its conjugates by incubating with strong base,followed by recovery of PSA, usually by adsorption and elution from ananion exchange column. The purified PSA with reduced endotoxin contentcan be used to derivatise drug delivery systems, biological molecules,including protein and peptide drugs, which improves the pharmacokineticsand pharmacodynamics of drugs.

Polysialic acids are naturally occurring unbranched polymers of sialicacid produced by certain bacterial strains and in mammals in certaincells [Roth et. al, 1993]. They can be produced in various degrees ofpolymerisation from n=about 80 or more sialic acid residues down to n=2by limited acid hydrolysis or by digestion with neuraminidases, or byfractionation of the natural, bacterially derived forms of the polymer.The composition of different polysialic acids also varies such thatthere are homopolymeric forms i.e. the alpha-2,8-linked polysialic acidcomprising the capsular polysaccharide of E. coli strain K1 and thegroup-B meningococci, which is also found on the embryonic form of theneuronal cell adhesion molecule (N-CAM). Heteropolymeric forms alsoexist—such as the alternating alpha-2,8 alpha-2,9 polysialic acid of E.coli strain K92 and group C polysaccharides of N. meningitidis. Sialicacid may also be found in alternating copolymers with monomers otherthan sialic acid such as group W135 or group Y of N. meningitidis.Polysialic acids have important biological functions including theevasion of the immune and complement systems by pathogenic bacteria andthe regulation of glial adhesiveness of immature neurons during foetaldevelopment (wherein the polymer has an anti-adhesive function)[Muhlenhoff et. al., 1998], although there are no known receptors forpolysialic acids in mammals. The alpha-2,8-linked polysialic acid of E.coli strain K1 is also known as ‘colominic acid (CA)’ and is used (invarious lengths) to exemplify the present invention.

The alpha-2,8 linked form of polysialic acid, among bacterialpolysaccharides, is non-immunogenic (eliciting neither T-cell orantibody responses in mammalian subjects, even when conjugated toimmunogenic carrier proteins) which may reflect its status as amammalian (as well as a bacterial) polymer. Shorter forms of the polymer(up to n=4) are found on cell-surface gangliosides, which are widelydistributed in the body, and are believed to effectively impose andmaintain immunological tolerance to polysialic acid. In recent years,the biological properties of polysialic acids, particularly those of thealpha-2,8 linked homopolymeric polysialic acid, have been exploited tomodify the pharmacokinetic properties of protein and low molecularweight drug molecules [Gregoriadis, 2006; Jain et. al., 2003; U.S. Pat.No. 5,846,951; WO-A-0187922]. Polysialic acid derivatisation gives riseto dramatic improvements in circulating half-life for a number oftherapeutic proteins including catalase and asparaginase, and alsoallows such proteins to be used in the face of pre-existing antibodiesraised as an undesirable (and sometimes inevitable) consequence of priorexposure to the therapeutic protein. In many respects, the modifiedproperties of polysialylated proteins are comparable to proteinsderivatised with polyethylene glycol (PEG). For example, in each case,half-lives are increased, and proteins and peptides are more stable toproteolytic digestion, but retention of biological activity appears tobe greater with

PSA than with PEG [Hreczuk-Hirst et. al., 2002]. Also, there arequestions about the use of PEG with therapeutic agents that have to beadministered chronically, since PEG is only very slowly biodegradable[Beranova et. al., 2000] and high molecular weight forms tend toaccumulate in the tissues [Bendele et. al., 1998; Convers et. al.,1997]. PEGylated proteins have been found to generate anti PEGantibodies that could also influence the residence time of the conjugatein the blood circulation [Cheng et. al., 1999]. Despite the establishedhistory of PEG as a parenterally administered polymer conjugated totherapeutics, a better understanding of its immunotoxicology,pharmacology and metabolism will be required [Hunter and Moghimi, 2002].Likewise there are concerns about the utility of PEG in therapeuticagents that may require high dosages, since accumulation of PEG may leadto toxicity. The alpha-2,8 linked polysialic acid therefore offers anattractive alternative to PEG, being an immunologically invisiblebiodegradable polymer which is naturally part of the human body, andwhich degrades, via tissue neuraminidases, to sialic acid, a non-toxicsaccharide. However the crude PSA is contaminated with high levels ofendotoxin which limits its therapeutic utility.

Our group has described, in previous scientific papers and in grantedpatents, purification and fractionation of PSA and its utility inimproving the pharmacokinetic properties of protein therapeutics[Gregoriadis, 2006, Jain et. al., 2004; US-A-05846,951; WO-A-0187922].Now, we describe preparation of purified

PSAs with reduced endotoxin content which can be used to producePSA-derivatised proteins, and other therapeutic agents. These newmaterials and methods are particularly suitable for the production ofPSA-derivatised therapeutic agents intended for use in humans andanimals, in which the chemical and molecular definition of drug entitiesis of major importance due to medical ethics and the safety requirementsof the regulatory authorities such as the FDA and EMEA.

Endotoxin (which is a lipopolysaccharide) was first defined by RichardPfeiffer in 1892 as a heat stable toxic substance released upondisruption of microbial envelopes. The inflammatory response in theinfected host results in the production of toxicity, which appears to beoptimally adapted for the clearance of most local infections. However,an inflammatory response leading to septic shock and death may alsooccur when there is systemic distribution of severe infections.

This lipopolysaccharide (LPS) is utilized in most of the steps thatoccur during the presentation of endotoxin to the myeloid cells of theimmune system and the production of inflammatory cytokines. The mostpotent component of LPS is lipid A and has become synonymous withendotoxin. Inflammatory mediators from bacteria like peptidoglycan, thediacylglycerylcysteine moiety of bacterial lipoproteins, and bacterialnucleic acid signatures, are also referred to as endotoxin. Recently, ithas been discovered that Toll-like receptor (TLR4) is the lipid Ainflammatory signal transducer. In addition, the signal transducers fordifferent inflammatory mediators have been identified. The structure ofendotoxin is important since its elucidation facilitates anunderstanding of how it can be removed.

LPS is made up of three parts: the proximal, hydrophobic lipid A region,which anchors LPS to the outer leaflet of the OM, the distal,hydrophilic O-antigen repeats, which extend into the aqueous medium, andthe interconnecting core oligosaccharide. The O-antigen and core sugarsalthough not essential for survival, provide bacterial resistanceagainst various antimicrobial agents including detergents and themembrane attack complex of serum complement.

Wild-type cells that produce O-antigen due to their glossy colony areknown as ‘smooth’ and those which do not have O-antigen are known as‘rough’. The molecules that contain the O-antigen polysaccharide areusually referred to as LPS and the molecules lacking O-antigen, as inNeisseria, are termed lipooligosaccharide or LOS. As Lipid A isessential for survival and is also a potent inflammatory mediator, it isnow a target for the development of antibiotics and anti-inflammatoryagents.

Many inflammatory mediators, in addition to LPS, can be regarded asendotoxins, including peptidoglycan, the diacyl glycerylcysteine moietyof bacterial lipoproteins, bacterial nucleic acid signatures andinactive endotoxins (produced by endotoxin mutant strains).

Modified lipid A structure has been used to develop novel endotoxinantagonists and immune adjuvants [Christ et al., 1995]. Determination ofthe biochemical details of lipid A structure and function helps tounderstand its role in bacterial pathogenesis and to intervene withnovel treatments for infection [Bishop 2005].

Methods have been developed to reduce the endotoxin content of fluids.WO87/07531, for instance, describes the removal of endotoxin fromendotoxin-containing fluids, such as blood, using polymyxin Bimmobilised on a solid phase support.

U.S. Pat. No. 6,942,802 B2 describes methods for removing bacterialendotoxin from a protein solution to recover protein. In this patent,immobilised metal affinity chromatography is used.

U.S. Pat. No. 6,713,611 B2 relates to a method of removing an endotoxinfrom a solution containing basic proteins. The method comprises adding asurfactant to the solution and loading the resultant solution onto acation exchange column with subsequent recovery of the basic proteinfrom the column.

U.S. Pat. No. 6,617,443 describes a method for removing endotoxins froma material from which nucleic acids are to be recovered. The endotoxinsare removed by pre-incubating the nucleic acids in a salt-free detergentsolution and subsequent anion exchange chromatography on a tentacleanion exchanger.

U.S. Pat. No. 5,169,535 describes a method for removing endotoxin from asolution containing protein and endotoxin, where the pH of the solutionis adjusted to pH 9 or lower (the isoelectric point of the protein) andthe solution is passed through a column packed with a cross-linkedgranular chitosan. The endotoxin is absorbed and the protein passesthrough.

U.S. Pat. No. 5,917,022 describe a process for the removal of endotoxinfrom a biological product such as proteins for therapeutic use, bloodplasma fractions, and albumin solutions. This method comprises ofbinding endotoxin present in the said biological product to across-linked hydrophilic matrix made up of copolymer of allyl dextranand N,N′-methylene bisacrylamide, to which the endotoxin binds with somedegree of specificity. This process is in the class of affinitychromatographic method.

U.S. Pat. No. 7,109,322 disclose a process for reducing or removingendotoxins from compositions containing therapeutic active substances,usually nucleic acids, extracted from natural sources by geneticengineering and/or biotechnology. For that purpose, the compositionsbeing recovered from fermentation broth, for instance, are treated withchromatographic materials. The fractions of natural sources such as E.coli fermentation broth obtained are centrifuged, lysed using 200 mMNaOH, 1% sodium dodecyl sulphate, filtered, then passed through an anionexchange column to which the nucleic acid preferentially (compared toendotoxin) absorbs. The nucleic acid is eluted with an elution bufferand recovered. In another example a metal affinity absorbent forendotoxin is used prior to the anion exchange column.

U.S. Pat. No. 6,699,386 B2 invention provides an absorbent having a highability to absorb endotoxin selectively and a method of adsorbingendotoxin from a protein solution. The adsorbent is comprised of a basicsubstance bonded to a base material by means of a cross-linking agent.

U.S. Pat. No. 6,774,102 invention describes blood treating materialhaving the capacity to selectively remove endotoxin and cytokineinducing substances from blood or plasma by extracorporeal adsorptionfor therapeutic septic shock treatment. They also provided methods anddevices which used an adsorbent having a polydisperse oligopeptide ofthe invention immobilized on a solid state support medium for removingendotoxin from the blood of human or animal subject.

Methods have also been described for the removal of endotoxin frompolysaccharide samples.

U.S. Pat. No. 5,589,591 discloses a process for producing asubstantially endotoxin-free polysaccharide composition using a sizeseparation technique, preferably ultrafiltration. The method is suitablefor removing endotoxin from mannan, gum arabic and arabinogalactanrecovered from their plant sources.

US 5,039,610 describes a process for removing endotoxin fromGram-negative polysaccharides such as polyribosylribitol phosphate.Polysaccharide-containing powder derived from Gram-negative bacteriafermentation broth is solubilised to provide a divalent counter ion forendotoxin. Alcohol is added incrementally to induce lipopolysaccharideprecipitation and the resultant material is mixed with a non-ionicresin, a detergent and a chelating agent.

However, to date, it remains a challenge to reduce the endotoxin contentof a polysialic acid sample to an acceptable level for use inpharmaceuticals. Polysaccharide from fermentation broths has an anioniccontent and molecular weight very similar to endotoxin. The naturalcompound is also a lipopolysaccharide. For these reasons mostpurification techniques result in some copurification of PSA andendotoxin. The present invention overcomes these problems.

In accordance with a first aspect of the invention, we provide a processfor reducing the endotoxin content of a sample containing polysialicacid and endotoxin comprising the sequential steps:

-   -   (i) adding to the sample a base having a pKa of at least 12 to        form a basic solution having a pH of at least 12, incubating the        solution for a pre-determined time at a pre-determined        temperature; and then    -   (ii) recovering polysialic acid having reduced endotoxin content        from the solution.    -   In one embodiment the recovery of PSA from the solution includes        the steps:    -   (iii) passing the base-treated solution through an        anion-exchange column whereby polysialic acid is absorbed on the        ion exchange resin;    -   (iv) washing the column with one washing buffer, whereby        polysialic acid remains absorbed on the ion exchange resin; and    -   (v) eluting the polysialic acid from the column using an elution        buffer to provide a product solution of polysialic acid having        reduced endotoxin content. It is believed that at least some of        the base-treated endotoxin product passes through the ion        exchange column in the void volume.

In another embodiment the recovery step (ii) involves a salt-removaltreatment step, i.e. based on size exclusion chromatography wherebyhigher molecular weight materials, such as polysaccharides are recoveredfree of salts. The invention is of particular utility for treatment ofmicrobial fermentation broths.

The polysialic acid and endotoxin containing solution which is subjectedto the base treatment of the process of the invention is preferablyfermentation broth, usually the supernatant after centrifugation, or theproduct of preliminary treatment steps for removal of unwantedcontaminants such as nutrients, lipids, proteins and nucleic acid. Thesample may, however, contain protein which is not wanted in the finalproduct as it is expected that this will be degraded by the base and thedegradation products removed easily during the PSA recovery steps.

This process results in samples of PSA which have reduced endotoxincontent and which are suitable for use in the preparation ofpharmaceuticals. Surprisingly, to the skilled person, the use of thestrong base does not result in degradation (of molecular weight) ordeacetylation of the PSA. The skilled person would have avoided contactof PSA with a strong base such as NaOH where deacetylation is to beavoided, see for instance Moe et al. We have shown, however, thatincubating with base followed by recovery of PSA eg by elution in ananion exchange column is a particularly effective method for reducingthe endotoxin content of PSA, while not leading to chemical modification(deacetylation, for instance), nor molecular weight reduction.

In accordance with a second aspect of the invention, there is provided asample of PSA material having reduced endotoxin content obtainable bythe above process.

The endotoxin content should be reduced to a pharmaceutically acceptablelevel. Preferably, the endotoxin content of the sample, after theprocess of the invention, is less than 25 EU/mg of PSA material.Accordingly, a third aspect of the invention provides a sample of PSAcomprising an endotoxin content of 25 EU/mg of PSA material, or less,measured using the LAL test. Preferably, the endotoxin content is in therange 0.05-25 EU/mg of PSA material. The present invention providessamples of PSA itself and PSA- conjugate (eg biological molecule, ordrug delivery system conjugate) (hereinafter referred to as PSAmaterial) that have a low endotoxin content.

The process of the invention is carried out under conditions thatprovide useful yields of PSA. It is possible to avoid contamination ofPSA with affinity ligands such as polymyxin B, or polycations such aspoly-D-lysine, that are commonly used in affinity adsorbents designed toremove endotoxin either by omitting the use of such materials completelyor using them under conditions at which no degradation of the matrixoccurs (under which endotoxin removal may not be optimised). This‘purified’ PSA may be used to derivatise therapeutic agents, such asproteins, and used in the human body without the risk of endotoxintoxicity.

The endotoxin level is measured using the LAL test as described inEuropean Pharmacopoeia 5.0, Appendix XIVC.

In step (i) of the process, the sample is added to a base having a pKaof at least 12. Without being bound by theory, it is thought that thebase cleaves bonds, probably ester bonds in the endotoxin, rendering itinactive, and/or allowing removal of the reaction products. To haveutility in the process of this invention the base must therefore besufficiently strong to react and hydrolyse the endotoxin. It issurprising that the endotoxin concentration reduced withoutsubstantially destroying the polysialic acid, for instance bydeacetylating the N-acetyl groups or cleaving the polysialic acidchains.

Preferably, the base has a pKa of at least 13, more preferably at least14. The base preferably forms a basic solution having a pH of at least13, more preferably at least 14. The sample is typically incubated inthe base in a suitable buffer, for instance HEPES buffer, or water.

Suitable bases for use in the present invention are NaOH, KOH, Ca(OH)₂and LiOH. Particularly preferred is NaOH. NaOH at a concentration of 2Nis suitable.

Step (i) is carried out for a pre-determined time which typically rangesfrom 5 minutes to 24 hours, preferably from 30 minutes to 6 hours. Thepre-determined temperature is typically in the range 0° C. or 60° C. andis preferably 2° C. to 40° C. Higher temperatures and/or longer timesmay lead to undesirable degradation of PSA itself.

In one embodiment of the invention, the incubation in step (i) iscarried out for 2 hours at 20° C. with constant stirring. Step (i) maybe immediately followed by an additional step in which the sample isneutralised. Neutralisation may be performed, for instance with HCl, toachieve a final solution pH of around 7.4.

A suitable method making use of ion exchange chromatography on whichsteps (iv) and (v) may be based is described in our earlier PatentApplication, WO2006/016161. This method can be adapted for use in thepresent invention, where it is not necessary to separate the PSA, atthis stage, into fractions of different average molecular weight. Inthat prior art method, polysialic acid is separated into fractions ofdifferent average molecular weight. Aqueous solution of PSA is contactedwith anion exchange resin in a column whereby PSA is adsorbed. The PSAis then subjected to selective elution by aqueous elution buffers, andthe PSA is recovered from eluted fractions. The selective elutioninvolves washing the resin in the column with at least one elutionbuffer. If more than one elution buffer is used, each may have differentionic strength and/or pH, in which the second and subsequent buffershave higher ionic strength and/or pH than the buffers of the immediatelypreceding step. The fractionation process using ion exchange leads to asmall reduction in endotoxin content.

In preferred embodiments of the process of this invention, the sample ispassed through an anion exchange column after having been treated withthe base. The sample may need to be prepared before loading onto thecolumn, for instance, it may need to be diluted. At least some of theendotoxin will pass through the column and it is preferred that theloading buffer is selected such that PSA is full adsorbed and at leastsome endotoxin fails to be adsorbed. Examples of loading buffers aredescribed below.

Step (iv) of the method is a washing step, in which a low ionicconcentration elution buffer is washed through the column. For instancesuch an initial wash step may be carried out with a buffer having a saltconcentration of 100 mM or less. Typically, the washing buffer comprisestriethanolamine and has a pH around 7.4. This initial step may wash outlow molecular weight contaminants. The washing step may involve a volumeof at least 1, preferably at least 1.5 column volumes, based on thevolume of the ion exchange resin column. During this step, substantiallyall of the polysialic acid remains on the ion exchange resin. More thanone washing step may be performed. The second washing buffer may beidentical to the first, or alternatively, comprise an alcohol and/or asurfactant. Suitable examples of surfactants are nonionics such as PEG,Tween 20 and 80 and Triton. The washing steps may remove furtherendotoxin from the column.

It is to be noted that a column of anion exchange resin may have adiameter of the cross-section which is greater than the height or may,as in a conventional column, have a height which is greater than thediameter of the cross section. The volume may be in the range 1 to 5000ml. The height of the column may be in the range 1 cm to 5000 cm. Thecross sectional area may be in the range 1 cm to 5000 cm². The crosssection may be of any shape but is preferably round.

After the anion-exchange resin washing step, the PSA is eluted usingelution buffer. The elution buffer used in step (v) generally has arelatively higher ionic strength than the washing buffer, in order toremove the PSA from the column. Typically, the elution buffer comprisesNaCl at a concentration of at least 0.4 M.

We have found that it is preferred for the elution step of the preferredembodiment to use at least 1.0, preferably at least 1.25 and mostpreferably at least 1.5 column volumes of the respective elution buffer.Preferably no more than 3 column volumes of elution buffer are used. Theflow rate for a 75 ml matrix is preferably 7 ml/minute.

The preferred embodiment of the process may comprise more than one, forinstance at least 5, for instance as many as 20 or, generally in therange 6 to 12, sequential steps of elution with elution buffers ofsuccessively increasing ionic strength. The ionic strength in the firstof these essential steps is generally in the range 1 mM to 1 M. In thismanner, eluted polysialic acid may be fractionated into samples havingdifferent average molecular weight.

Preferably the ionic strength of an elution buffer is varied by varyingthe level of a salt of a strong mineral acid and a strong mineral base,preferably sodium chloride.

After step (v) has been performed, and the product solution obtained,steps (i) and (iii) to (v) may be repeated. Preferably, steps (iii)-(v)are repeated.

The PSA may be further treated. Subsequent steps may include furtherpurification and/or concentration steps. With regard to further steps,these generally involve steps in which the polysaccharide is isolatedfrom any salt, for instance using membranes, for instanceultra-filtration membranes. Such steps may additionally allowconcentration of the PSAs to form more highly concentrated solutions.Such solutions may be subjected to additional steps of membranetreatment, for instance successive ultra-filtration or other filtrationsteps.

The anion-exchange elution buffer may contain a volatile acid or baseand in this case the further steps may involve volatilisation of thevolatile base from the eluted fractions. Although it is possible torecover the PSA from aqueous solutions by precipitation techniques, forinstance involving non solvents for the PSAs, it is preferred that nosuch solvents are utilised, since this may make final isolation from therespective solvents more difficult. Consequently the final step ofrecovery preferably involves evaporation of the water and, preferably,any remaining volatile buffer components remaining from the elutionsteps. In a preferred case wherein triethanolamine is present, both thetriethanolamine cation and acetate anion are volatile and can easily beremoved under vacuum.

The PSA may be finally isolated from solution by drying, preferablyunder reduced pressure. This is preferably performed by freeze-drying.

Precipitation, preferably using a non solvent, may be carried out as apreliminary step to fractionation to remove a portion of the populationand decrease polydispersity of the higher molecular weight fractions.Preferably, differential ethanol precipitation is used.

The process may comprise an additional step, either before step (i),between steps (i) and (ii) or after step (ii). This may involvehydrophobic interaction chromatography (HIC), affinity chromatography,such as immobilised metal affinity chromatography (IMAC) or sizeexclusion chromatography (SEC).

A suitable HIC column is one to which endotoxin is adsorbed but to whichPSA does not adsorb, such as Phenyl FF. The loading buffer is typicallyammonium sulphate, sodium chloride etc. in deionised water, or deionisedwater adjusted to pH 7.4.

HIC is preferably carried out before step (i). In fact, it is believedthat this is the first disclosure of use of HIC for the removal ofendotoxin from a sample of PSA. It is surprising that HIC can be used toremove endotoxin without co-removal of polysialic acid such as recoveredfrom fermentation broth, i.e. in the form of a lipopolysaccharide, orvice versa, for instance where PSA material is conjugated to render itmore hydrophobic than endotoxin. By appropriate selection of column andconditions endotoxin will adsorb to the column while PSA material passesthrough or or vice versa, either into the void column (with loadingbuffer) or by eluting with suitable elution buffer. In accordance with afurther aspect of the invention we therefore provide a novel process forreducing the endotoxin content of a sample containing polysialic acidand endotoxin comprising passing the sample through a hydrophobicinteraction column to which endotoxin adsorbs, whereby PSA passesthrough the column and is collected to provide a product solution ofpolysialic acid having reduced endotoxin content. According to a yetfurther aspect of the invention we provide a novel process for reducingthe endotoxin content of a sample containing polysialic acid andendotoxin comprising passing the sample through a hydrophobicinteraction column to which the PSA material adsorbs, whereby endotoxinpasses through the column and PSA material is adsorbed, and then PSAmaterial is eluted and collected to provide a product solution ofpolysialic acid having reduced endotoxin content. The use of a phenylcolumn is preferred, as this binds to endotoxin whilst allowing PSA topass through the column. In the first embodiment, after passage of thevoid volume containing endotoxin, the column may be washed with awashing buffer and washing fractions collected, which may also comprisePSA. The second embodiment is of particular utility for purifying PSAconjugates with proteins, such as relatively hydrophobic proteins.

Affinity filtration media may be used in the process of the presentinvention usually after step (ii). Affinity matrices are selected whichspecifically trap endotoxin. Conditions are selected such that PSA isnot adsorbed when the solution is loaded. It may often be preferred toavoid use of endotoxin-specific affinity chromatography, to avoidcontamination by degradation products of the matrix. Preferably, whereany such process step is included, the conditions are selected to avoidsuch degradation. Although such conditions may be selected at theexpense of optimising endotoxin removal, when combined with theessential steps of the process of the invention in a multistep processthe endotoxin level can be reduced to acceptable levels

In one embodiment of the present invention, affinity matrix comprisingan immobilised endotoxin-binding agent is used to purify the PSA sample.A suitable agent is a polymyxin B gel, in particular Detoxi-Gellm™.

The Detoxi-Gel™ Endotoxin Removing Gel uses immobilized polymyxin B tobind and remove pyrogens from solution. The polymyxins are a family ofantibiotics that contain a cationic cyclopeptide with a fatty acidchain. Polymyxin B neutralizes the biological activity of endotoxins bybinding to the lipid A portion of bacterial lipopolysaccharide. Studiesperformed by Kluger et. al,(1985) indicate that the immobilizedpolymyxin B inactivates some but not all endotoxins.

The immobilized polymyxin B gel is a stable affinity matrix that resistsleaching of ligand into the valuable preparation. Making use of anaffinity support permits easy cleanup of buffers, cell culture media,solutions containing macromolecules such as proteins, andpharmacologically important components. Detoxi-Gel™ Endotoxin RemovingGel also has been used to remove endotoxin from nucleic acid (DNA)samples (Wicks et. al., 1995).

Typically, before a polymyxin B gel is used, it must first be degassed.Gels may be regenerated by washing, typically with a detergent, forinstance sodium deoxycholate, followed by washing to remove thedetergent. A suitable agent to wash the gel is pyrogen-free water. Oncegenerated, the gel is applied to the column and pyrogen free buffer, orwater, are added. The column incubation time is typically one hour. Oncethe sample has been collected, it is typically lyophilized to preventbacterial contamination.

A Cellufine™ column is another type of affinity chromatography column,that may be used to reduce the endotoxin content of the PSA sample. Sucha column is typically equilibrated with 2-8, for instance 5 columnvolumes of endotoxin free buffer, for instance HEPES buffer (pH 7.4).HEPES may also be used as the loading buffer. Cellufine is aparticularly suitable material to use as it has low non-specific binderof PSA.

Other commercially available endotoxin-selective affinity chromatographycolumns are the EndoTrap columns. An EndoTrap Red™ column can also beused to reduce the endotoxin content of the PSA sample. Such a column istypically equilibrated with 5-10, for instance 5 column volumes ofendotoxin free buffer, for instance with Regeneration Buffer Red™ and 20mM HEPES buffer (pH 7.4) or with deionised water (pH 7.4). HEPES ordeionized water (pH 7.4) may also be used as the loading buffer.

An EndoTrap Blue™ column may be used to reduce the endotoxin content ofthe PSA sample. Such a column is typically equilibrated with 5-10, forinstance 5 column volumes of deionised water (pH 7.4), supplemented with50-100 μM Ca⁺².

In the process according to the first aspect of the invention, thesample may be subjected to additional purification means. For instance,the sample may be subjected to one or more preliminary steps ofincubation with a surfactant, a chelating agent, a base, an organicsolvent, an oxidant or a peroxidase, before step (i), between steps (i)and (ii) or after step (ii).

Preferably, the surfactant is an anionic surfactant, for example, sodiumdodecyl sulphate (SDS). Alternatively, the surfactant may be 0.5% TritonX 114 or 1% Triton X 100 or 114. Alternatively cationic surfactants,e.g. bactericidally-effective surface active compounds such ascetyltrimethylammonium, bromide may be used. A suitable incubation timeis 1 hour. Temperatures in the range 0-50° C. are suitable, although35-37° C. is preferred.

Where a nonionic surfactant is used the solution is loaded onto an anionexchange column for removal of nonionic surfactants. Here, 20 mM TEAbuffer can be used.

In one embodiment of the present invention, the PSA sample is incubatedwith 1% SDS/2N NaOH in HEPES buffer for 1 hour, and then the solution isloaded onto a column to remove salt and anionic surfactant, forinstance, a GPC column, and fractionated with a suitable buffer,typically HEPES buffer. A pd10, Sephadex 25 column may be used.

Alternatively, other nonionic surfactants having different cloud pointsto Triton, such as Tween 20 and Tween 80, can also be used to reduceendotoxin content of the PSA sample. The PSA sample is incubated in 0.1%Tween 80 for 30 minutes at room temperature. Then the solution is loadedonto an anion exchange column with a suitable buffer or deionised water(pH 7.4) for removal of surfactants. The column is washed extensivelywith water and buffer (pH 7.4) to remove surfactant.

In embodiments the process includes an oxidation step. This step isintended to oxidise the end unit of polysialic acid to render itreactive, e.g. with proteins or peptides. It may simultaneously lead toa reduction in endotoxin content and thus be a useful step to achievethe overall objective of endotoxin reduction. Suitable oxidants aresodium periodate, superoxide, hypochlorite and peroxidase.

The process of the invention may comprise use of phase extraction,diafiltration precipitation and/or use of dry heat before step (i) orafter step (ii). When dry heat is used, typically the PSA sample isheated in a vial to a temperature in the range 100-200° C., mostpreferably around 150° C. for 1-6 hours, typically around 4 hours. Theseconditions do not deactivate or degrade PSA.

Diafiltration may be used subsequent to incubation of the PSA samplewith a surfactant, or alternatively, used independently of surfactanttreatment. The filter should be small enough to retain at least highermolecular weight PSA whilst allowing endotoxin to pass through. Atypical ultrafiltration apparatus with a cutoff of 5 kDa for globularproteins has pores sufficiently large to allow passage of endotoxin(˜10,000 Da) whilst retaining PSAs of ˜20,000 and above. A diafiltrationstep is preferably used after the base treatment, e.g. as part ofrecovery step (ii).

The process of the present invention provides PSA with reduced endotoxincontent to pharmaceutically acceptable levels, for human or veterinaryuse. Ideally, the endotoxin content of the product PSA is less than 25EU/mg PSA. Preferably, the endotoxin content of the PSA is in the range0.05-25 EU/mg.

The present invention may be of utility for producing PSA conjugateshaving low endotoxin content. In a PSA conjugate preferably theconjugated moiety is preferably a biological molecule, more preferably aprotein. PSA protein conjugates may be produced by a variety of methods,in particular see our previous Patent Applications WO-A-0187922 andWO92/22331. The process according to the first aspect of the inventionis generally unsuitable for PSA-biological molecule conjugates since thebase typically damages the biological moiety, i.e. any conjugation isnot carried out prior to step (i). In a fourth aspect of the inventionthere is provided a process comprising the sequential steps:

-   -   (v) PSA conjugate—endotoxin mixture is contacted with a nonionic        surfactant for a predetermined time at a predetermined        temperature;    -   (vi) the surfactant-treated sample is passed through an        anion-exchange column whereby the PSA conjugate is adsorbed on        the column;    -   (vii) the PSA conjugate is eluted from the column using an        elution buffer to produce a PSA conjugate solution having        reduced endotoxin content.

Examples of nonionic surfactant to be used in this aspect are forinstance PEG compounds, sorbitan monooleate Tween 20/80 or a member ofthe series Triton. Again, without wishing to be bound by theory, it isbelieved that the surfactant reduces endotoxin levels by disruptingendotoxin micelle formation, or by dissolving endotoxin.

The product of the fourth or first aspect of the invention may be apharmaceutical composition, in which case, the endotoxin content shouldbe low enough to avoid toxic side effects when the composition isadministered to a human or animal. The composition may be a human orveterinary pharmaceutical composition.

Typically, PSA samples, before purification, i.e. the starting materialfor step (i) of the first aspect and step (v) of the fourth aspect havean endotoxin content in the range 1000-200,000 EU/mg. In order to bepharmaceutically acceptable, the final endotoxin content of the samplemust be no more than 25 EU/mg. The permissible endotoxin content isdependent upon the intended use of the PSA. If the PSA is to be used toderivatise a protein to be used as a medicament, the permissibleendotoxin content varies with the dose of protein to be used as amedicament—higher doses typically require more stringent removal ofendotoxin.

For PSA-protein conjugates used in pharmaceutical compositions with ahuman (veterinary) dose of 10-500 mg, the endotoxin content should be nomore than 0.5 EU/mg, and is preferably in the range 0.05-0.5 EU/mg. ForPSA-protein conjugates with a human dose of 1-10 mg, the endotoxincontent should be no more than 5 EU/mg, and is preferably in the range0.5-5 EU/mg. For PSA-protein conjugates with a human dose of up to 1000μg, the endotoxin content should be no more than 25 EU/mg, and ispreferably in the range 5 to 25 EU/mg.

In the invention sample containing endotoxin and PSA is conveniently afermentation broth. The fermentation broth may be produced recombinantlyor be naturally occurring PSA-producing microbial broth, a hydrolysisproduct thereof or a fractionalised derivative of either of these. Themicrobes may be E coli K1, Neisseria meningitidis, or Moraxellaliquefaciens. Typically, the process according to the first aspect ofthe invention comprises a preliminary step in which the microbes arefermented to produce a fermentation broth. The PSA may be apoly(2,8-linked sialic acid), poly(2,9-linked sialic acid) or analternating 2,8- 2,9-linked PSA. Preferably, the PSA is colominic acid(CA) or an oxidised, reduced, aminated and/or hydrazide derivativethereof.

The PSA typically has at least 2, preferably at least 5, more preferablyat least 10, for instance at least 50 saccharide units. Typically, PSAsof most utility have a weight average molecular weight of up to 100 kDa.

The PSA may be derived from any source, preferably a natural source suchas a bacterial source, e.g. E. coil K1 or K92, group B meningococci, oreven cow's milk or N-CAM. The sialic acid polymer may be aheteropolymeric polymer such as group 135 or group V of N. meningitidis,or may be synthesized e.g. enzymatically. The PSA may be a blockcopolymer, such as a conjugate of a homo poly(sialic acid) with a blockof another naturally occurring polymer or synthetic polymer. The PSA maybe in the form of a salt or the free acid. It may be in a hydrolysedform, such that the molecular weight has been reduced following recoveryfrom a bacterial source. The PSA in the starting material for a processof the invention may be material having a wide spread of molecularweights such as having a polydispersity of more than 1.3, for instanceas much as 2 or more. Preferably the polydispersity of molecular weightis less than 1.2, more preferably less than 1.1, for instance as low as1.01.

The derivatisation of proteins and drug delivery systems with thepurified PSA may result in increased half life, improved stability,reduced immunogenicity, and/or control of solubility and hencebioavailability and pharmacokinetic properties, or may enhancesolubility actives or viscosity of solutions containing the derivatisedactive.

Preferably the PSAs of the final product of the process aspects of theinvention and of the new products have 2-1000 sialic acid units, forinstance 10-500, more preferably 10 to 50 sialic acid units. Preferably,the polydispersity of the PSA will be less than 2, ideally less than1.2, and ideally in the range 1.01 to 1.10.

The purification methods used in the present invention advantageouslyreduce the polydispersity of the PSA, in addition to reducing theendotoxin content.

The endotoxin content of a sample of PSA is reduced by the process ofthe invention by at least 5-fold, preferably at least 10-fold, mostpreferably at least 100, 200, 500 fold and in some embodiments up to1000, 10,000, 100,000 or even 1 million fold. Preferably the basetreatment and recovery reduces the endotoxin content at least 5-fold.The overall reduction by preliminary steps and multiple PSA-recoverysteps may lead to a reduction of at least 10⁵ fold. Generally thecombination of recovery steps is selected such that endotoxin reductionis maximised while PSA recovery is maximised by the use of steps whichare complementary to one another in terms of endotoxin fraction removal.

The “Assay for Endotoxin” (LAL test) section of the Examples describeshow the endotoxin content may be measured.

EXAMPLES Reference Assay for Endotoxin

To perform the assay for endotoxin, The Endosafe—PTS (Portable TestSystem) from Charles River Laboratories was used. This is based on theLAL (Limulus Amoebocyte Lysate) assay.

Instrument Operation

All required information was entered into the reader. Once all the testinformation had been entered, the reader displayed “add sample; pressenter”. The PSA samples were prepared, unless otherwise specified at 1mg/ml in 20 mM TEA buffer at pH 7.4. 25 μL of sample was pipetted intoall four sample reservoirs and enter key was pressed on the reader. Thepump drew sample aliquots into the test channel and the results wereproduced in 15-20 minutes. When the test was completed the instrumentdisplayed the endotoxin measurement and the assay acceptance criteria onthe screen. The instrument gave the following specifications: SampleEU/mL, sample % CV, spike EU/mL, spike % CV and % spike recovery.

Example 1 Endotoxin Reduction Using Sodium hydroxide

6 mg/ml solution of colominic acid contaminated with endotoxin (31 kDaunoxidised) was prepared in 0.5M NaOH Hepes buffer and was incubated atroom temperature for 10 minutes. Then 0.5 ml solution was loaded onsize-exclusion chromatography desalting column and the fractioncollected was discarded. The column was then washed with 2.5 ml HEPESbuffer and the fraction was collected followed by collection of another2 ml fraction with HEPES buffer. The elution fractions collected werethen analysed for colominic acid content by resorcinol assay. Thefractions containing colominic acid were then pooled together and wereanalysed for endotoxin content. The samples were tested for degree ofdeacetylation of the PSA. FIG. 1 shows the Native PAGE of colominic acidin 0.5 M NaOH and 1% SDS HEPES buffer solution.

There was a 53-fold reduction in endotoxin content using NaOH and therewas no detectable degree of deacetylation. Nor is any breakdown of CAobserved in the PAGE using NaOH and SDS.

Example 2 Reduction of Endotoxin by Anion Exchange After Base

A sample taken directly from E. coli K1 fermentor (1 mg/ml ) in 20 mMTEA buffer (pH 7.4) was measured and found to be more than 10⁵ EU/mg.Then NaOH was added to the PSA solution to make final normality of 2NNaOH in PSA sample solution and then it was incubated for 2 hours atroom temperature with gentle mixing. The pH of the solution wasrecorded. The HiTrap QFF (1 ml) column was washed with 10 column volumedeionised water (pH 7.4) and then column was equilibrated using 10column volumes of 20 mM TEA. The conductivity of the sample solution wasmeasured and was diluted appropriately with 20 mM TEA buffer to matchthe conductivity of buffer solution. The sample solution was then loadedon QFF (1 ml column) at the rate of 1 ml/mim and the void volume (˜⅓column volume) was collected separately. 1 ml fractions were collected.The column was then washed with 20 mM TEA and washing fractions of 1 mleach was collected. The sample was then eluted with 1 M NaCl in 20 mMTEA and the fractions were collected. Resorcinol assay of the elutionsamples was performed to calculate the amount of colominic acid presentin the samples and the endotoxin content of the pooled elution samplescontaining colominic acid was determined. The endotoxin level of theproduct was found to be reduced to 1407 EU/mg, i.e. more than ˜71 fold.The recovery of PSA was 91%.

The base treatment and recovery by anion exchange was repeated takingabove product and further reduction in endotoxin content was seen, downto less than 300, i.e. a further 5-fold reduction.

Example 3 Reduction of Endotoxin by Anion Exchange After NonionicSurfactant Treatment (Triton X-100)

Endotoxin content of the original sample (1 mg/ml) in 20 mM TEA buffer(pH 7.4) was measured. Colominic acid solution (35 mg/ml contaminatedwith endotoxin) was prepared in 1% Triton X 100 and was incubated for 2hours at room temperature. The pH of the solution was measured. TheHiTrap QFF column was prepared as in Example 2 and the sample wasprepared and loaded as in Example 2. The column was then washed with 20mM TEA and washing fractions of 1 ml each were collected. The sample wasthen eluted with 1 M NaCl in 20 mM TEA and the fractions were collected.Resorcinol assay of the elution samples was performed to calculate theamount of colominic acid present in the samples and the endotoxincontent of the pooled elution samples containing colominic acid wasdetermined. Where the loading of colominic acid was 7.28 mg, theendotoxin content of the starting material was reduced from 4023 to 1511EU/mg, i.e. about 3 fold. Recovery was 97%.

Example 4 Reduction of Endotoxin by Nonionic Surfactant Treatment(Triton X 114) and Anion Exchange

1% Triton X 114 solution was prepared and was added to PSA solutionderived from fermentation broth by standard centrifugation, lysis,diafiltration ad ultrafiltration in appropriate amount to make the finalconcentration of 0.5% Triton X 114 and was incubated for 2 hours at roomtemperature. The pH of the solution was measured. The HiTrap QFF columnwas prepared, loaded, washed and eluted as in Example 3 and theendotoxin content of the pooled elution samples containing colominicacid was determined. The starting endotoxin level of more than 10⁵ EU/mgwas reduced to around 2.3×10⁴ EU/mg, i.e. by around 4 fold.

Example 5 Reduction in Endotoxin by Base/Anion Exchange Followed byNonionic Surfactant and Anion Exchange

Colominic acid solution product of a different fermentation broth wastreated by base by the process of Example 2 and then treated with TritonX 114 as below, then anion exchange.

1% Triton X 114 solution was prepared and was added to PSA solution inappropriate amount to make the final concentration of 0.5% Triton X 114.The solution becomes cloudy at room temperature (25° C.). To make thesolution clear, it was kept in ice for 10-15 mins. Again the solutionwas kept at room temperature for 20 mins to make it cloudy. The cloudysolution was centrifuged and two layers were separated: upper layercontaining PSA and lower layer of Triton X 114 containing endotoxin. Theupper layer was kept to load on to the QFF column. The HiTrap QFF columnwas prepared, loaded, washed and eluted as in Example 4.

In this case the level of endotoxin was reduced from over 10⁵ to around4.4 to 10³ EU/mg in the first base treatment step and further to 8.2×10²EU/mg after the second surfactant step and anion exchange.

Example 6 Reduction of Endotoxin by Nonionic Surfactant Treatment (Tween80) and Anion Exchange

Colominic acid solution (100 mg/ml contaminated with endotoxin; 2.5 gbatch) was prepared in 0.1% Tween 80 and the pH was adjusted to 7.4. Thesolution was incubated for 30 minutes at room temperature and diluted to500 ml with water pH 7.4. The HiTrap QFF column (75 ml) was washed with10 column volume deionised water (pH 7.4) and then the column wasequilibrated using 10 column volumes of water pH 7.4. The samplesolution was then loaded at the rate of 7 ml/min at room temperature.The loading fractions were collected in fractions of 50 ml falcon or asappropriate. The first 25 ml of loading out was collected separatelywhich accounts for the void volume of the column. Column was then washedwith 0.01% Tween 80 in water pH 7.4 (7 ml/min, 4 CV) and washingfractions of 75 ml each were collected. The column was then washed withwater pH 7.4 (7 ml/min, 4 CV) and collected the washing fractions of 75ml or as appropriate. The column was then washed with 150 mM sodiumchloride in water pH 7.4 (7 ml/min, CV) and collected the fractions of75 ml or as appropriate. The sample was then eluted with 500 mM sodiumchloride in water pH 7.4 and collected the elution fraction of 75 ml oras appropriate. The samples were collected at room temperature.Resorcinol assay of the elution samples was performed to calculate theamount of colominic acid present in the samples and the endotoxincontent of the pooled elution samples containing colominic acid wasdetermined.

Example 7 Endotoxin Removal by Hydrophobic Interaction Chromatography

2 M Ammonium sulphate in deionised water/deionised water was used as theloading buffer. 500 μg of Colominic Acid (7 kDa produced in ReferenceExample 1) was dissolved in 500 μL of loading buffer and the solutionwas loaded on the HIC columns specified in the Table below. The columnswere then incubated for one hour. Elution samples were collected andresorcinol assay was performed to estimate the amount of colominic acidin the elution samples. Elution samples containing colominic acid werepooled and analysed for endotoxin content.

Results Original Final Endotoxin Endotoxin Column Loading ColominicContent Content Used Buffer Acid (μg) (EU/mg) (EU/mg) Butyl FF 2M(NH₄)₂SO₄ 336.95 3070 211 Phenyl FF 2M (NH₄)₂SO₄ 353.80 3070 148 OctylFF Deionised 379.49 3070 214 Water

Endotoxin content was reduced by up to 14.5 fold. The order of Endotoxinreduction by different columns were in order: Phenyl>Butyl≧Octyl.

Example 8 Endotoxin Removal Using Anionic Surfactant (Sodium DodecylSulphate)

6 mg/ml solution of colominic Acid (31 KDa contaminated with endotoxin)was prepared in 1% SDS HEPES Buffer and was incubated for 1 hour at 37°C. Then 0.5 ml solution was loaded on Pd10 column and the fractioncollected was discarded. The column was then washed with 2.5 ml HEPESbuffer and the fractions were collected. The column was then eluted with2 ml HEPES buffer. The elution fractions collected were then analysedfor colominic acid content by resorcinol assay. The fractions containingcolominic acid were then pooled together and were analysed for endotoxincontent.

Endotoxin content was reduced from >10exp6 EU/mg to 741.6 EU/mg. Therecovery of the PSA was 84%.

Reference Example 1 Fractionation Using Anion Exchange

A new prepacked column (1000 ml; Q Sepharose FF, GE Healthcare) wasprepared and the preservative was washed with three column volumes ofdeionised water, and then with 3 column volumes of wash buffer at a flowrate of 50 ml/min. The pump tubing was filled with start buffer(triethanolamine buffer, pH 7.4; 20 mM) and the column was connected tothe pump and a few drops of start buffer were applied to the top of thecolumn to avoid introducing air into the column. Colominic acid solutionderived from E. coli fermentation broth contaminated with endotoxin wasprepared in triethanolamine buffer and the pH of the solution wasadjusted to 7.4. The sample (colominic acid (CA) was obtained fromMarukin, Japan) 750 ml in wash buffer) was then applied to column at therate 50 ml/min, followed by washing of the column with another 750 ml ofwash buffer. The column was then washed with 1500 ml washing buffer. Thebound CA was eluted with 1500 ml of different elution buffers from 100mM NaCl to 475 mM NaCl collecting washings from each run andtransferring them in respective containers. All residual CA and otherresidues were removed with 1500 ml 1 M NaCl and the washings werecollected. The column was then regenerated with 3 column volumes of washbuffer. The column was then stored in 20% ethanol at RT. The large chainlength samples (removed by the high salt eluent) were then concentratedto minimum volume in 250 ml concentrators (Vivacell, Vivascience) under4 bar pressure at 4° C. The concentrates were washed four times withdistilled water (pH 7.4 adjusted with NaOH). The small chain lengthsamples were also concentrated in 50 ml concentrators (Vivaflow,Vivascience) under pressure at 4° C. The concentrate was washed fourtimes with distilled water. Samples were assayed for colominic acidcontent by resorcinol assay. The samples were then analyzed forendotoxin content.

The results show that the endotoxin level was reduced from a value of1.6×10exp5 EU/mg to 3070 EU/mg. There was almost a 5 fold reduction inthe endotoxin content.

Reference Example 2 Affinity Chromatography—Detoxi Gel ColumnPurification

The Detoxi gel columns were regenerated using pyrogen free solutions toprevent introducing any endotoxin into the sample. All the solutionswere degassed before applying to the column to prevent air bubbles fromclogging the column and reducing the flow. Detoxi-gel endotoxin removinggel may be used at least 10 times without loss of activity. All thesolutions and gel were equilibrated to room temperature before use.

The gels were degassed by placing slurry in the bottom of a suctionfilter flask with a magnetic stirrer. While the slurry was stirred anaspirator was used to create a vacuum within the flask. The gel wasdegassed for approximately 15 minutes. The appropriate sized column waspacked with degassed slurry and the gel was allowed to settle down for30 minutes. The gels were regenerated by washing with five columnvolumes of 1% sodium deoxycholate, followed by 3-5 column volumes of apyrogen free water to remove excess. The gels were regenerated beforeeach use following the same procedure. The sample was then applied tothe column. Aliquots of pyrogen free buffer or water were added and theflow through was collected. The sample emerged from the column after thevoid volume collection was completed (94% of the bed volume). Forgreater efficiency, the bottom and top caps were replaced after samplehas entered the gel bed. The column was incubated for at least one hour,the top and bottom caps were removed sequentially. Pyrogen free bufferor water was then added to collect the samples. Caution was taken toprevent sample contamination from dust or dirty glassware subsequent toendotoxin removal in all these experiments. Samples were then frozen andstored. The columns were regenerated following the same procedure asabove to remove any bound endotoxin and were stored in 25% ethanol at2-8° C.

In the first example, the sample is the product of a process asdisclosed in Example 3 of WO2008/012525, of colominic acid fractionatedby the techniques described in WO2006/016161 conjugated to GCSF. Theconjugate prior to affinity gel treatment had an endotoxin level of 438EU/mg, and after treatment the level was reduced to 10.5 EU/mg.Endotoxin content of PSA-protein conjugates was reduced up to 35 timesusing this affinity chromatography material.

In a second example the sample was neat colominic acid used under thefollowing conditions:

PSA (mg/ml) [Resorcinol Formulation assay] Amount of PSA (mg) Vol pH PSA19.3 KDa 0.1749 0.1574 0.9 7.4 (Marukin) In Hepes buffer (20 mM mlHepes, 150 mM NaCl; pH 7.4)

Endotoxin content was reduced to 4.2 EU/mg from original value of 16,000EU/mg. The recovery was more than >90%.

In a third exemplification the starting material was the solutionproduced in Reference Example 1, under the following conditions. Theresults are shown below:

PSA (mg/ml) Endotoxin [Resorcinol Amount of Content Formulation assay]PSA (mg) Volume (EU/mg) CA 7 kDa 0.374 1.87 5 ml 66.04 CA 7 kDa 0.1600.8 5 ml 178.75

Original endotoxin content in the sample was 3070 EU/mg. The reductionin the endotoxin content was up to 47 fold.

CONCLUSION

Endotoxin-specific affinity chromatography may be used to removeendotoxin from polysialic acid and conjugates thereof, i.e. we haveshown that it is possible to select conditions under which endotoxinbinds to the column while PSA does not bind but can instead be recoveredin a convenient form and with very low endotoxin level. The step maythus be useful to treat PSA products of base (or surfactant) treatment.

Reference Example 3 Endotoxin Removal Using Affinity Column (Cellufine)

This Example is similar to Reference Example 2 but uses a differentendotoxin-removing affinity column. Cellufine ET clean column (S beads)was regenerated by washing with 5 column volumes of 0.2 M NaOH, 2M NaCland then with endotoxin free water. The column was then equilibratedwith 5 column volumes of suitable endotoxin free buffer (HEPES Buffer).The colominic acid solution produced in Reference Example 1 in HEPESbuffer (1 mg/ml) was then applied to the column at a flow rate of0.1-0.2 ml/min. at 21° C. The total amount loaded was 218 μg. The columnwas then incubated for one hour and then the elutions were collectedwith HEPES buffer. Resorcinol assay of the elution samples was performedto calculate the amount of colominic acid present in the samples and theendotoxin content of the pooled elution samples containing colominicacid was determined.

The endotoxin level was reduced from 3070 to 75 EU/mg i.e. amount40-fold.

The method was repeated using the GCSF-PSA conjugate used as thestarting material for Reference Example 2 part 1, reducing the endotoxinlevel from 438 to 12.4 EU/mg.

We conclude that the Cellufine column is suitable for removing endotoxinfrom PSA.

Reference Example 4 Removal of Endotoxin Affinity Column (EndoTrap Red)

This example uses another affinity column for endotoxin adsorption.Endotrap Red column was regenerated and equilibrated using deionisedwater pH 7.4. 10 ml of 50 mg/ml colominic acid solution contaminationwith endotoxin was prepared. Two columns were kept in series. The samplesolution was loaded onto the column and the void volume was collected(˜1/3 of the column volume). Then remaining loading solution wascollected. Then the column was washed with 6 column volume deionisedwater pH 7.4 and the fractions of 1 ml each was collected. Resorcinolassay for all the fractions was performed to calculate the amount ofcolominic acid present and the endotoxin content of the samplescontaining colominic acid was determined.

The results for 16 kDa colominic acid show a reduction in endotoxinlevel from 564 to 6 EU/mg, a reduction of around 90 fold.

The procedure was also used for an endotoxin-contaminated insulin-PSAconjugate produced by the method described in WO2008/012528. Theendotoxin content was reduced from 111 to 12.5 EU/mg, a 9-foldreduction.

These examples show that another column is useful for removal ofendotoxin from PSA and conjugates thereof.

Reference Example 5 Removal of Endotoxin Through Affinity Column UsingHEPES Buffer

Endotrap Red column (1 ml column volume) was regenerated withregeneration Buffer Red provided with the column. The column was thenequilibrated using 5 column volumes of suitable endotoxin free 20 mMHEPES Buffer. The endotoxin-contaminated PSA solution in HEPES buffer(750 μg/ml) was then applied to the column at a flow rate of 0.1-0.2ml/min at 21° C. Then the column was eluted using 0.3 ml of 20 mM HEPESbuffer. Resorcinol/protein assay of the elution samples was performed tocalculate the amount of colominic acid present in the samples and theendotoxin content of the pooled eluted samples containing colominic acidwas determined. The results show that the endotoxin level may be reducedby around 2 to 7 fold, and that this is affected by the load of PSA used(lower loads on the column give better reductions) at loading levels atwhich 100 PSA recovery is achieved.

Reference Example Removal of Endotoxin, Proteins, DNA and Cell DebrisThrough Anion Exchange Column Using 30% IPA

Fermentation broth recovered by centrifugation, supernatant recovery,diafiltration and ultrafiltration, with a colominic acid concentrationof 35 mg/ml was prepared and its pH was measured. The HiTrap QFF columnwas washed with 10 column volume deionised water (pH 7.4) and column wasequilibrated using 10 column volumes of 20 mM TEA. The conductivity ofthe sample solution was measured and was diluted appropriately with 20mM TEA buffer to match the conductivity of buffer solution. The samplesolution was then loaded at the rate of 1 ml/min fractions werecollected separately. Then 1 ml of fractions was collected. Column wasthen washed with 30% IPA and washing fractions of 1 ml each wascollected. The sample was then eluted with 1 M NaCl in 20 mM TEA and thefractions were collected. Resorcinol assay of the elution samples wasperformed to calculate the amount of colominic acid present in thesamples and the endotoxin content of the pooled elution samplescontaining colominic acid was determined. The initial endotoxin contentor the broth extract was 3.2×10⁴ EU/mg and the final content was 1.8×10³EU/mg, i.e. the endotoxin content was reduced by 18 fold.

REFERENCES

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1. A process for reducing the endotoxin content of a sample containingpolysialic acid (PSA) and endotoxin comprising the steps of: (i) addingto the sample a base having a pKa of at least 12 to form a basicsolution having a pH of at least 12, (ii) incubating the solution for apredetermined time at a pre-determined temperature; and (iii) recoveringpolysialic acid having reduced endotoxin content.
 2. The process ofclaim 1 wherein the base has a pKa of at least
 13. 3. The process ofclaim 1 wherein the pH of the said basic solution is at least
 13. 4. Theprocess of claim 1 wherein the base is NaOH, KOH, Ca(OH)₂ or LiOH. 5.The process of claim 4 wherein the base is 2N NaOH.
 6. The process ofclaim 1, wherein the pre-determined temperature is in the range 0 to 60°C.
 7. The process of claim 1 in which step (iii) includes the followingsequential substeps: (a) passing the sample through an anion-exchangecolumn whereby polysialic acid is adsorbed on the ion exchange resin;(b) washing the column with a washing buffer, whereby polysialic acidremains adsorbed on the ion exchange resin; and (c) eluting thepolysialic acid from the column using an elution buffer to provide aproduct solution of polysialic acid having reduced endotoxin content. 8.The process of claim 7, wherein the sample, after step (ii), isneutralised before performing step (iii).
 9. The process of claim 7,wherein in step (b), the column is washed in a first wash buffer of lowionic strength to elute endotoxin from the column and wherein theelution buffer used in step (c) has relatively higher ionic strength.10. The process of claim 9, wherein the washing buffer and/or theelution buffer contains volatile base.
 11. The process of claim 9,wherein a second wash buffer comprises NaCl at a concentration of atleast 0.2M.
 12. The process of claim 7, wherein in step (b) the columnis washed more than once.
 13. The process of claim 7, wherein steps (i)and (iii) are repeated on the product solution.
 14. The process of claim1 comprising at least one further step in which the endotoxin level ofpolysialic acid is reduced by purification carried out either beforestep (i), between steps (ii) and (iii) or after step (iii) wherein saidstep is selected from ion exchange chromatography, hydrophobicinteraction chromatography, affinity chromatography, size exclusionchromatography and combinations thereof.
 15. The process of claim 14,wherein hydrophobic interaction chromatography is carried out beforestep (i).
 16. The process of claim 1 wherein the sample is incubatedwith a surfactant, a chelating agent, an organic solvent, an oxidant ora peroxidase either before step (i), between steps (ii) and (iii) orafter step (iii).
 17. The process of claim 1, further comprisingobtaining said sample by fermenting E. coli to produce a fermentationbroth containing the PSA and endotoxin, and optionally removing protein,lipid, nucleic acid and/or nutrients, to form said sample.
 18. Theprocess of claim 1 wherein the endotoxin content of the PSA in theproduct solution is no more than 25 EU/mg of PSA, measured by the LALtest.
 19. A sample of fermentation broth having reduced endotoxincontent obtainable by a process according to claim
 1. 20. A sample ofpolysialic acid (PSA) comprising an endotoxin content suitable forveterinary or human administration.
 21. A sample according to claim 20,having an endotoxin content of no more than 25 EU/mg of PSA.
 22. Asample according to claim 20, wherein the PSA is a poly(2,8-linkedsialic acid), a poly(2,9-linked sialic acid), or alternating 2,8-2,9-linked polysialic acid, preferably poly (2,8-linked sialic acid). 23.A sample according to claim 22, wherein the PSA is colominic acid (CA),or an oxidised, reduced, aminated and/or hydrazide derivative thereof.24. The process of claim 1, wherein the predetermined time ranges from 5minutes to 24 hours.
 25. The process of claim 24, wherein thepredetermined time ranges from 30 minutes to 6 hours.
 26. The process ofclaim 1 wherein the sample contains polysialic acid-protein conjugateand reduced endotoxin content.
 27. The process of claim 26 wherein thereduced endotoxin content is no more than 5 EU/mg.
 28. The process ofclaim 27 wherein the reduced endotoxin content is no more than 0.5EU/mg.
 29. A process for preparing a polysialic acid-biological moleculeconjugate, comprising reducing the endotoxin content of a samplecontaining polysialic acid according to the process of claim 1 andconjugating the polysialic acid obtained with a biological molecule. 30.The process of claim 29, wherein the biological molecule is a protein.